Toxoplasma gondii is a ubiquitous intracellular parasite with felids as definitive hosts and a broad range of intermediate hosts. Rodents are considered suitable sentinels for prevalence studies of many infections, including toxoplasmosis. This study aimed to estimate the seroprevalence of T. gondii in rodents from different localities of Slovakia and investigate the correlation between the seropositivity and the species, age, sex, and sexual activity of animals. Altogether, 1009 wild rodents belonging to 9 species were trapped in 2015 and 2019, and antibodies to T. gondii were detected in 6.7% of the animals. Seropositivity was detected in seven species, ranging from 0.0% in Micromys minutus and Apodemus sylvaticus to 7.7% in A. flavicollis. The females reached significantly higher seropositivity (9.7%) than the males (3.8%), and the adults were positive significantly more often (9.2%) than the subadults (4.9%). The seropositivity differed also among localities, with significantly higher positivity detected in suburban and touristic areas (12.2%) than in localities with a lower level of human activities (5.5%). This study showed that the occurrence of T. gondii varies significantly in rodent species and habitats with various environmental conditions and different levels of anthropic use. Several biological and ecological factors, e.g., soil contamination, soil conditions, the susceptibility of rodent species etc., may influence this variability.